Navigation Links
Transformation of Nematodes With the Helios Gene Gun

Peter Jackstadt1, Horst Zahner2 and Gerd Hobom1, 1 Institut fr Mikrobiologie und Molekularbiologie der Justus-Liebig Universitt Gieen, Frankfurter Str. 107, D-35392, Gieen, 2 Institut fr Parasitologie der Justus-Liebig-Universitt Gieen, Rudolf-Buchheim-Str. 2, D-35392 Gieen

The Biolistic DNA transfer technique was first established for plant cells and plant tissues (Sandford et al., 1987; Jefferson et al., 1987; Bruce et al., 1989; Armaleo et al., 1990). More recent applications have been DNA transfer experiments aiming at DNA vaccination of animals (genetic immunization; Sundaram et al., 1996), for example a bombardment of mice to generate protective antibodies, instead of the DNA injection used for this purpose in the past.

Here we report on the potential to use this convenient method for transfection of nematodes, not only for the free-living soil nematode Caenorhabditis elegans, the well known model organism in its class, but also for the cotton rat filaria Litomosoides sigmodontis, which serves as model for human pathogenic filariae (Zahner, Hobom and Stirm, 1995), such as Wuchereria bancrofti or Brugia spp. These parasitic species cause a severe lymphatic disease called filariasis (elephantiasis), from which more than 100 million human beings suffer worldwide.

C. elegans is cultivated on NGM agar plates (Brenner, 1974). Wild-type worms (strain N2 var. Bristol) are fed on Escherichia coli OP-50 (exogenous uracil dependent mutant) seeded on those plates. The surface of a densely populated NGM agar plate is subsequently rinsed with tap water. A drop of water containing nematodes is placed in the center of a 6 cm Petri dish on ice (to reduce their motility). In this state C. elegans could be used for microparticle bombardment (Figure 1A).

For the same purpose adult females of L. sigmodontis must be isolated from the pleural cavity of their laboratory host Mastomys coucha (Wegerhof and Zahner, 1985), and stored in RPMI medium (supplemented with 10% FCS, Gibco-BRL; 1% Penicillin and Streptomycin). Single parasites are transferred to an agar plate and used for microprojectile bombardment.

DNA-coated gold particles are chosen as microparticles following a modified Bio-Rad protocol for the Helios gene gun. The protocol is as follows:
1. Mix 50 mg of gold microparticles (Bio-Rad; a 1:1 mixture of particles 0.6 /1.0 in diameter) with 100 l of 0.05 M spermidine (Sigma), and sonicate 3 times for 3 seconds in an Eppendorf tube (1.5 ml).

2. Add 200 l of purified supercoiled plasmid DNA to the carrier suspension (in a total volume of 100 l). It is possible to use more than one plasmid simultaneously.

3. CaCl2 (100 l, 1 M) is now added dropwise with continuous vortexing. Precipitation was allowed to take place for 10 minutes at room temperature.

4. Wash the suspension three times with 1 ml of absolute ethanol (Sigma-Aldrich), and pellet the sample by centrifugation (12,000 rpm for 1 minute, Heraeus Biofuge).

5. Finally, the pellet is resuspended in 3.5 ml of ethanol containing 0.1 mg/ml polyvinylpyrrolidone (PVP; MW 360,000). In this state a DNA-coated gold particle suspension can be stored for up to 2 months at -20 C (if not directly used for cartridge preparation).

1. Clean the inner surface of the Tefzel tubing (external size 10 mm; internal diameter 6 mm; Bio-Rad) with 5 ml of absolute ethanol and dry with nitrogen (grade 4.8; 99.998% pure) in the rotating tubing holder for at least 5 minutes.

2. Sonicate the DNA-coated particle suspension in ethanol for 3 seconds (to achieve a homogeneous solution) and load it into the tubing at a rate of 5.56 ml/min using a syringe.

3. Allow the DNA-coated particles to settle for 3 minutes.

4. Remove the ethanol solution quickly (within 30 seconds).

5. For drying the wall-attached particles, flush nitrogen gas through the tubing for 3 minutes at a flow rate of 0.35 l/min.

6. Finally, cut the resulting particle-covered tubing into individual cartridges 16 mm in length (one full length of tubing results in nearly 50 cartridges) to fit into the 12-shot barrels of the Helios gene gun (Bio-Rad). See Figure 2.

7. In a desiccated atmosphere, these can be stored at 4 C for up to 8 months without losing activity.

8. Deliver the DNA-coated gold particles using 4 blasts of helium (grade 4.5; 99.995% pure) applied to the cartridges at a pressure of 300 p.s.i. and a distance of 3 cm to the target (in the case of C. elegans).

9. A pressure of 120180 p.s.i. at a distance of 2 cm using 1 blast of helium is useful for the transfer into parasitic nematode L. sigmodontis. After this procedure, the female parasites may be cultivated at 37 C for up to 2 weeks in RPMI medium (5% CO2).

While the majority (up to 90%) of C. elegans hermaphrodites are killed during Biolistic DNA transfer, the survivors with their gold particles incorporated may be cultivated on new E. coli-seeded NGM agar plates (Figure 2A shows particles within a living hermaphrodite). F1/F2 progeny nematodes were screened 3 to 4 days later for visible dominant mutant marker rol-6 (Kramer et al., 1990 ). Animals carrying roller allele su-1006 exhibit a helically twisted cuticle and body. Muscle contractions, which would normally propel the worm forward or backward in a sweeping sinusoidal motion, will cause the nematode instead to roll over its longitudinal axis and to move in circles (Mello et al., 1991; Fire, 1986; Kramer et al., 1990). In the case of L. sigmodontis, no special selection mode is necessary because the success of bombardment can be visualized by standard light microscopy to make visible the gold particles now located within these filariae (see Figure 2B).

The established mode of DNA transfer into C. elegans is microinjection of DNA into the gonadal syncytium (Stinchcomb et al., 1985; Fire, 1986). Under standard selection conditions promoter-reporter gene cDNA constructs are used, in co-transfection with a visible dominant such as mutation rol-6 (su1006), which leads to roller movement phenotype of transformed worms (already in F1 generation) and is easily detectable within a background of normally moving animals. The same transfection efficiency has been observed for this Biolistic DNA transfer method as for microinjection experiments (Fire, 1986), while this new strategy is less time consuming and much more convenient for co-transfection experiments. In the F1 generation, DNA transfer rates of up to 1% were observed using this selection method, which became considerably enriched during following generations after further transfer (F3/F4: 20%; F5/F6 up to 90%), proving their genetic fixation.

Besides marker gene rol-6, we have employed two different reporter genes, a mutant form of GFP (green fluorescent protein), which was originally cloned from the jellyfish Aequorea victoria (Prasher et al., 1992; Cody et al., 1993; Chalfie et al., 1994) with enhanced fluorescence (Siemering et al., 1996), and -galactosidase from Escherichia coli. Expression was visualized by standard fluorescence microscopy (GFP; see Figure 4) or by histochemical staining (lacZ).

In both cases we have observed marker gene expression under transcriptional control of the SV40 early promoter (Figure 3 A and B), or of the C. elegans actin-1 core promoter after Helios gene gun mediated delivery of Au/DNA particles into the nematode. The lacZ expression of constructs containing the actin-1 core promoter of C. elegans (Files et al., 1983; Krause et al., 1989; Stone and Shaw, 1993) in DNA transfected L. sigmodontis will even be analyzed histochemically.

The Biolistic DNA-transfection procedure, because of its simple mode of handling, has an important potential in various innovative molecular biology applications, such as DNA vaccination, protein targeting, and basic research applications such as gene regulation studies. The new Biolistic technology should therefore be able to substitute for other applications in this field. Also, the prepared DNA/Au particles can be stored for rather long periods, at least up to 8 months, which makes repetitions or comparative experiments more meaningful. In applying this technique to various nematodes, it is conveniently possible to use a couple of hundred target worms of C. elegans and proceed in comparatively low precision, while DNA injections have to be done individually and precisely into the gonads. Parasitic worms, however, can only be transfected on an individual basis or in small groups by Biolistic gene transfer. In all experiments designed, reporter gene expression is observed independent from distance of initial gold particle target sites, clearly indicating an DNA drift within the entire bodies. Altogether, this technique is a reproducible and efficient method of producing fertile transgenic nematodes.

1. Armaleo, D., Ye, G.-N., Klein, T. M., Shark, K. B., Sandford, J. C. and Johnston, S. A., Curr. Genet., 17, 97103 (1990).

2. Brenner, S., Genetics, 77, 7194 (1974).

3. Bruce, W. B., Christensen, A. H., Klein, T., Fromm, M. and Quail, P. H., PNAS, 86, 96929696 (1989).

4. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C., Science, 263, 802804 (1994).

5. Cody C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G. and Ward, W. W., Biochemistry, 32, 12121218 (1992).

6. Files, J. G., Carr, S. and Hirsh, D., J. Mol. Biol., 164, 355375 (1983).

7. Fire, A., EMBO J., 5, 26732680 (1986).

8. Franz, M. and Andrews, P., Z. Parasitenkd., 72, 387395 (1986).

9. Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W., EMBO J., 6, 39013907 (1987).

10. Kramer, J. M., French, R. P., Park, E. and Johnson, J. J., Mol. Cell Biol., 10, 20812090 (1990).

11. Krause, M., Wild, M., Rosenzweig, B. and Hirsh, D., J. Mol. Biol., 208, 381392 (1989).

12. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. and Cormier, M. J., Gene, 111, 229233 (1992).

13. Sandford, J. C., Klein, T. M., Wolf, E. D. and Allen, N., Part. Sci. Technol., 5, 2737 (1987).

14. Siemering, K. R., Golbik, R., Sever, R. and Haseloff, J., Current Biology, 6, 16531663 (1996).

15. Stinchcomb, D., Shaw, J. E., Carr, S. H. and Hirsh, D., Mol. Cell Biol., 5, 34843496 (1985).

16. Stones, S. and Shaw, J. E., Gene, 131, 167173 (1993).

17. Sundaram, P., Xiao, W. and Brandsma, J. L., Nucleic Acids Research, 7, 13751377 (1996).

18. Zahner, H. and Wegerhof, P. H., Z. Parasitenkd., 71, 583593 (1985).

19. Zahner, H., Hobom, G. and Stirm, S., Parasitology Today (Review), 11, 116120 (1995).

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) via Sonderforschungsbereich 535. We thank Noelia Pohl, Ulrike Ruppert and Brigitte Hofmann for their exceptional technical assistance. Many thanks to PD Dr. R. Dennis for helpful discussion and comments on the manuscript.

Biolistic is a registered trademark of E. I. duPont de Nemours and Co.
Biolistic technology is exclusively licensed to Bio-Rad Laboratories.
Eppendorf is a trademark of Eppendorf Geratebau Netheler + Hinz, GmbH.

back to top


Page: All 1 2 3 4 5 6 7

Related biology technology :

1. Highest Possible Transformation Efficiencies for High-Throughput Applications
2. New Competent Cells for Highest Transformation Efficiencies
3. Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System
4. Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells
5. Transformation of Filamentous Fungi by Microprojectile Bombardment
6. Transformation of Filamentous Fungi by High-Voltage Electroporation
7. Delivery of pCMV-S DNA Using the Helios Gene Gun System Is Superior to Intramuscular Injection in Balb/c Mice
8. Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun
9. Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment
Post Your Comments:
TAG: Transformation Nematodes With the Helios Gene Gun

(Date:7/29/2015)... 2015  AsureQuality and Ubiquitome have agreed to ... in food and primary production sectors. The collaboration ... device, the Freedom4. AsureQuality provides quality ... supermarket shelf for producers, processors and Competent Authorities ... disease control and pest management and surveillance programmes. ...
(Date:7/29/2015)... , July 29, 2015 ... has announced the addition of the "Global ... their offering. The Global Biosimilars Market, ... of the rapidly growing biosimilars market. With the ... striving to minimize costs, biosimilars are being viewed ...
(Date:7/29/2015)...  AmnioChor Inc., an early stage biotech startup based ... announce that the Musculoskeletal Transplant Foundation of ... their seed round of development of the proprietary AmnioCept™ ... AmnioChor,s technology allows cryopreservation of the amniotic ... living within those tissues. Amnion is a well-established source ...
(Date:7/29/2015)... 29, 2015  Pfenex Inc. (NYSE MKT: PFNX) announced ... be released on Thursday, August 13, 2015, before the ... management will host a conference call to discuss the ... release outlining the financial results and business update will ... Please call 1-866-376-8058 (US) or 1-412-542-4131 (international) and reference ...
Breaking Biology Technology:AsureQuality and Ubiquitome Partner on Applications for Mobile Molecular Food Safety Testing 2Global Biosimilars Market to 2025: Monoclonal Antibodies (mAbs), Insulin, Interferons, G-CSF/ GM-CSF, Erythropoietins and Others 2Global Biosimilars Market to 2025: Monoclonal Antibodies (mAbs), Insulin, Interferons, G-CSF/ GM-CSF, Erythropoietins and Others 3The Musculoskeletal Transplant Foundation Agrees to Invest in AmnioChor 2Pfenex To Report Second Quarter 2015 Results and Provide Business Update on Thursday, August 13, 2015 2
... 15, 2011 /PRNewswire-Asia/ -- ShangPharma Corporation (NYSE: ... leading China-based pharmaceutical and biotechnology research and development ... ("Hengrui"), a leading Chinese pharmaceutical company, today announced ... of ShangPharma, and Hengrui have entered into a ...
... 2011 EvaluatePharma Ltd, the research company, is ...  With a team based in Tokyo, EvaluatePharma Japan KK will ... Japan. "Japan is a very important market for ... major customers from a distance, but it is now time ...
... RICHMOND, Calif., Sept. 14, 2011 Sangamo BioSciences, Inc. ... Lanphier, Sangamo,s president and CEO, will provide an overview ... on Wednesday, September 21, 2011 at the UBS 2011 ... New York City. In addition, Geoffrey Nichol, ...
Cached Biology Technology:ShangPharma Subsidiary and Jiangsu Hengrui Medicine Partner in Therapeutic Antibody Research and Development 2ShangPharma Subsidiary and Jiangsu Hengrui Medicine Partner in Therapeutic Antibody Research and Development 3EvaluatePharma Japan KK - Now Open for Business in Tokyo 2Sangamo BioSciences Announces Presentation at the 2011 UBS Global Life Sciences Conference 2
(Date:7/27/2015)... , July 27, 2015  Synaptics Inc. ... human interface solutions, today announced that Huawei has ... touchscreen solution for its stylish smartwatch. Huawei chose ... proven reliability, low power and highly responsive human ... finger. Huawei designers also required a classic round ...
(Date:7/21/2015)... July 21, 2015 Today, ZTE announced its ... as well as expected revenues in 2015 that relate to sales ... revenue guidance of approximately 2,200 MSEK for 2015. Jörgen ... leading smartphone manufacturer in China and we ... 02 5 for Axon , ...
(Date:7/13/2015)... July 13, 2015 Synaptics Inc. (NASDAQ: ... interface solutions, today announced sampling of ClearPad ® ... driver integration (TDDI) product targeting smartphones and tablets. ... combine Synaptics , best-in-class touch controller IP ... technology developed in the company,s Japan Design Center. ...
Breaking Biology News(10 mins):Synaptics Touchscreen Solution Powers New Huawei Watch 2Synaptics Touchscreen Solution Powers New Huawei Watch 3FPC's Touch Fingerprint Sensor FPC1025 in ZTE's Smartphone Axon 2Synaptics Announces Sampling of Second Generation TDDI Solutions 2Synaptics Announces Sampling of Second Generation TDDI Solutions 3
... Ind. -- Modern conservation techniques have brought us ... harvests and the rescue of prized lobster fisheries. ... wrought such failures, from the catastrophic loss of ... kill in 2006" In this week,s special ...
... (from plants or animals) that we consume on a daily ... pesticides or antibiotics. A doctoral thesis carried out by Jorge ... Department of Analytical Chemistry , at the University of Granada ... by professors Ana Mara Garc a Campaa and Laura G ...
... began more than 50 years ago as a way ... University of Tennessee researcher to a significant finding about ... about conservation. In the middle of the 20th ... bait introduced a new species of salamander to California ...
Cached Biology News:Why conservation efforts often fail 2Alternative methods proposed to detect pesticides and antibiotics in water and natural food 2UT researcher sheds new light on hybrid animals 2UT researcher sheds new light on hybrid animals 3
BD BioCoat Fibronectin 35 mm Culture Dishes, tissue-culture treated polystyrene with a uniform application of human fibronectin....
... DNA Kit provides a positive selection for ... protein expression in insect cells. The kit ... recombinant baculoviruses by eliminating the time-consuming steps ... an AcNPV genome with a portion of ...
... uses include large-scale colony-counting and plaque-lifting, microbial ... TM automated colony picking and gridding ... product number, created to easily match Cornings ... please order under the old Sigma-Aldrich number ...
Mouse monoclonal [7B9] to beta II Tubulin ( Abpromise for all tested applications). Antigen: Synthetic peptide, CEEEEGEDEA found at the c-terminus of beta II tubulin. Entrez GeneID: 10383 ...
Biology Products: